Abstract
Cherry leaf spot (CLS), caused by Passalora circumscissa, is a fungal disease that can cause decreased fruit quality and yield via inconsistent ripening or premature defoliation. Germplasm resource screening is the most reliable approach to disease control for economically important crops. However, information is limited in China about the resistance of cherry cultivars to leaf spot caused by P. circumscissa. The aim of this study was to identify the resistance levels of cherry cultivars. Fifty-two cherry cultivars, including 40 Prunus avium, four Prunus pseudocerasus, and eight Prunus cerasus cultivars were collected for resistance level characterization. These specimens were then used to screen for P. circumscissa resistance through both detached leaf assays and natural field infection. Significant differences in the disease index (DI) value among test cultivars, ranging from 5.7 (resistant) to 53.7 (highly susceptible) and 6.5 (resistant) to 53.2 (highly susceptible), were observed under the controlled and field conditions respectively. Correlation coefficients between DI in pairs of years were highly significant (0.77–0.86). Although resistance rankings for cherry cultivars between screening methods were variable, the resistance levels of 52 cultivars evaluated under controlled and field condition were comparable with a correlation coefficient of 0.70 (P < 0.01). The results indicated that, across cherry cultivars, responses to CLS in the detached leaf assay corresponded well to responses under field conditions. A detached leaf assay was developed as a complementary method to facilitate the screening of cherry cultivars for resistance to leaf spot caused by P. circumscissa. Our study provides a theoretical basis for cherry disease resistance breeding and rational cultivar utilization.
Cherry fruit are rich in vitamin C and polyphenols, exhibiting antioxidant and anti-inflammatory properties (Cubero et al., 2010; Garrido et al., 2012; Kelley et al., 2018; McCune et al., 2010). However, commercial cherry production is constrained by fungal and bacterial diseases that damage or kill these trees—particularly CLS, which has been detected in major cherry-producing countries including Chile, Turkey, Italy, and the United States (Guo et al., 2018; Iličić et al., 2018; Joshua and Mmbaga, 2015). CLS and related diseases can cause major economic losses (Wharton et al., 2003), making their prevention a priority.
Cherry production in China has risen significantly during the past decade. China now has both the largest total sweet cherry cultivation area (200,000 hm2) and the greatest annual sweet cherry yield (≈1,000,000 t) in the world (Li et al., 2019). These increases have coincided with the rising prevalence of cherry foliar diseases in many regions of China (Zhao and Liu, 2012; Zong et al., 2015). Leaf spot caused by Passalora circumscissa, has arisen as a disease of cherry trees that poses a threat to sweet and sour cherry production in China (Sun et al., 2017). Passalora circumscissa (syn. Cercospora circumscissa Sacc.; Teleomorph Mycosphaerella cerasella Aderh.) was first detected by Saccado in Italy and France in 1878. More recently, this pathogen has been detected in Germany and the United States, and it was first detected in China in the 1980s by Guo and Liu (1989). It impacts primarily the leaves of cherry trees, resulting in early chlorosis and premature defoliation during the summer. This defoliation leads these trees to be more susceptible to cold-induced injury and death during the winter, and also results in major reductions (≈40%) in fruit yield and fruit quality, with some variability in these outcomes based on the cherry cultivar, environmental conditions, and field management strategies (Pirnia et al., 2012; Sztejnberg, 1986).
Under cold conditions, P. circumscissa can attach to dead organs on cherry trees. During the spring, this leads to the development of sexual ascospores that can be spread readily by rain, which washes them onto nascent cherry leaves and subsequently infects them via their stomata (Jenkins, 1930; López-Carbonell et al., 1998). Conidial delivery to additional leaves and trees then occurs, spreading the disease rapidly (Verma and Gupta, 1979). Chemical control strategies are generally the most effective means of preventing this pathogen, but such efforts are often contraindicated because of their environmental impact and tendency to drive the development of fungicide resistance (Akhtar and Haq, 2003; Ma et al., 2006; Outwater et al., 2019; Proffer et al., 2006).
An appealing alternative to the chemical control strategy is the identification of cherry cultivars that are resistant to foliar disease, thereby protecting cherry yields in a cost-effective and environmentally friendly manner (Santi et al., 2004). Effective screening strategies are therefore needed to identify CLS-tolerant or -resistant germplasm resources (Tivoli et al., 2006). Some prior reports have described the use of artificial inoculation or field observation strategies to identify specific cherry cultivars that are resistant to certain bacteria and fungi (Psedomonas syringae pv. syringae, Pseudomonas syringae pv. morsprunorum, Blumeriella jaapi) (Andersen et al., 2018; Grzyb and Rozpara, 2004; Schuster and Tobutt, 2004; Wharton et al., 2003). Currently, however, little information is available regarding cherry cultivars that are resistant to CLS caused by P. circumscissa. Although not currently considered a yield-limiting factor in most areas of the world, P. circumscissa leaf spot disease on cherries is of increasing concern and is associated with a field incidence as high as 90% in some cultivation areas in China (Zhang et al., 2010).
In our study, therefore, we used multiple approaches to evaluate 52 commercial cherry cultivars to screen for P. circumscissa resistance.
Materials and Methods
Plant materials.
All cherry cultivars were planted during Spring 2011 in an experimental station at the Shandong Institute of Pomology, Taian, Shandong Province, China. These cultivars were grafted onto Gisela 148-1, with trees in the orchard being planted in a 2- × 4-m spacing scheme. No fungicides were applied to these trees, but all other standard recommended orchard, pest, and weed management strategies were used. In total, we evaluated 52 cherry cultivars for P. circumscissa CLS resistance, including four Chinese cherry (Prunus pseudocerasus), eight sour cherry (Prunus cerasus), and 40 sweet cherry (Prunus avium) cultivars.
Fungal isolation and identification.
Cherry leaves with typical leaf spot symptoms (leaf spot amphigenous, irregular to subcircular, 1 to 4 mm in diameter, scattered, sometimes coalescent, forming larger pale-brown to brown patches with a distinct border that is slightly raised and concolorous or darker brown) were collected from an orchard in Taian, Shandong Province, China, and transported to the laboratory in plastic bags and stored at 4 °C in a container. An improved single-spore isolation method was conducted based on a protocol outlined by Sun et al. (2019). Briefly, symptomatic diseased cherry leaves were first selected from the orchard, sterilized with 75% ethanol for 10 to 15 s, and rinsed three times with deionized water. A self-made acquisition tool was then used to isolate a single spore that was absorbed from the diseased spot, after which this spore was transferred onto the surface of a section of a 3% water agar plate. Individual germinated spores were then excised from these agar plates using a self-made cutting tool and a microscope under ×10 objective magnification. Last, germinated spores from these water agar plates were transferred onto potato dextrose agar media for growth and preservation. Fungal isolates were cultured on potato dextrose agar at 25 °C in the dark and verified as P. circumscissa via polymerase chain reaction (PCR). DNA was extracted from fungal mycelia by using a modified protocol published by Crous et al. (2009), and PCR was conducted using ITS1-F/ITS4-R (White et al., 1990) and EF-688F/EF1–1251R (Alves et al., 2008) primer pairs. PCR amplification and sequencing was conducted as described by Zhang et al. (2009). A BLAST search in GenBank using the ITS and EF-1α sequences revealed 100% identity with both sequences of P. circumscissa (KT428056, KX853061).
Inoculum preparation.
Inoculations were performed using mycelial fragment suspensions developed by Guo et al. (2018) and Kumakech et al. (2015). Before sporulation, mycelia were scraped, and the collected fragments were ground for ≈2 min in sterile distilled water using a pestle. The resultant solution was then filtered through two cheesecloth layers before mixing and adding a single drop of Tween-80. The concentration of this suspension was then evaluated with a hemocytometer and was adjusted to 1 × 106 mycelial fragments/mL using sterile distilled water. Individual drops of 1% Triton X-100 were added to these suspensions before inoculation to ensure the mycelia were able to adhere to the surfaces of infected leaves.
Detached leaf assay.
To assess the resistance of detached leaves from different cherry cultivars to P. circumscissa CLS, leaves of similar size and age from all cultivars were collected in June from an orchard in Taian. These leaves were then sterilized using 70% ethanol for 10 to 15 s, after which they were rinsed three times using sterile distilled water and then dried using sterile filter paper. Mycelial fragment suspensions were prepared and resuspended at 1 × 106 mycelial fragments/mL before inoculation. Leaves were then inoculated using a mist sprayer to apply this suspension to the entire surface of each leaf, followed by incubation for 48 h at 20 to 23 °C without light. The DI of these cultivars was evaluated via visual inspection and disease rating calculation after 14 d of inoculation, with this assay being repeated twice during two separate years.
Five healthy, uniformly shaped mature leaves from each cherry cultivar were used for another inoculation assay. Leaf inoculation was conducted by adding 5 μL of the mycelial fragment suspension (1 × 106 mycelial fragment/mL) to each site of infection on the upper leaf surface. These leaves were then placed in plastic boxes, covered with plastic film, and incubated at 25 °C while protected from light. Leaves were sprayed twice daily with sterile distilled water to ensure that humid conditions were maintained. Inoculation was conducted at three sites per leaf, whereas a fourth site was inoculated with sterile distilled water as a control. At 10 d postinoculation, a Vernier caliper, accurate to 0.1 mm, was used to measure mean lesion length (d) for all inoculated sites.
Field observation.
The rainy season in Tainan extends from June to October (average annual rainfall, 500–800 mm). Under these conditions, P. circumscissa CLS symptoms typically first appear during late June before peaking in late July to mid-August. We evaluated all trees for P. circumscissa CLS resistance during late August from 2013 to 2015, because disease symptoms were fully developed at this time. In total, we collected 200 randomly selected leaves from each cultivar to assess infected leaf areas.
Disease rating.
DI values were then used to separate these cherry cultivars into the following resistance categories: immune, DI = 0; highly resistant, DI = 0.1 to 5.0; resistant, DI = 5.1 to 15.0; moderately resistant, DI = 15.1 to 25.0; susceptible, DI = 25.1 to 35.0; or highly susceptible, DI = 35.1 to 65.0.
Statistical analysis.
Statistical Analysis System software 9.1 (SAS Institute, Cary, NC) was used for all statistical comparisons of susceptibility indices among P. circumscissa cherry cultivars over time.
Results
Field observation.
We observed significant differences among these 52 different cherry cultivars to their CLS resistance in field experiments (P < 0.05). Average DI values for these cultivars ranged from 6.5 to 53.2 across the 3-year study, indicating that none were immune or highly resistant to this disease. Eighteen of 52 accessions were found to be resistant (average DI, 10.88), whereas 16 accessions were moderately resistant (average DI, 19.63). Ten accessions and eight accessions were classified as susceptible and highly susceptible (average DIs, 28.54 and 42.25, respectively). These different resistance levels accounted for 35%, 31%, 19%, and 15% of total cultivars, respectively (Table 1).
Evaluation of cherry cultivar resistance to Passalora circumscissa leaf spot.
Detached leaf assay.
Next, detached leaf assays were used to categorize these cherry cultivars based on their DI. Through this approach, we found 13 (25%) cultivars to be resistant, 14 (27%) to be moderately resistant, 14 (27%) to be susceptible, and 11 (21%) to be highly susceptible to P. circumscissa, with no plants being immune or highly resistant to this fungal infection. The average DI values in these four susceptible categories were 9.71, 20.03, 29.55, and 42.25, respectively (Table 1).
Detached leaf assay DI values were higher than expected based on field observations. The cultivars SYT1502 and Chongqingwupi exhibited resistance in field observations, but were found to be susceptible in detached leaf assays. This may indicate there was not sufficient environmental stress in the field test to induce disease, and these cultivars are only relatively disease resistant.
We observed clear differences in lesion length among 52 different cherry cultivars following in vitro infection with P. circumscissa. The mean lesion length was 0.73 cm, and ranged from 0.15 cm for ‘Zaohongzhu’ to 1.22 cm for ‘Bigarreav Moreav’ (Table 1).
Correlation analyses.
Significant correlations between DI values were observed over the 3-year study period, indicating there was consistency from year to year in this analysis for all tested cultivars, and also highlighting a significant interaction between cultivars and years (Table 2).
Correlations between annual Passalora circumscissa cherry leaf spot field observation results.
Observations from 2013, 2014, and 2015, and overall average values were compared with matched cultivar results from the detached leaf assay (Table 3). We observed significant correlations between field observations and detached leaf spot test results (correlation coefficient range, 0.65–0.70), with differences among these cultivars indicating the extent to which the resistance of individual cultivars varied under field conditions. The observed strong correlation (r = 0.70, P < 0.01) between detached leaf spot assay results and the averages of field test findings over these 3 years indicates that comparisons of these two assays are highly reliable.
Correlations between detached leaf spot assay results and field observation findings pertaining to Passalora circumscissa cherry leaf spot resistance.
Significant differences were observed among cultivars, and a significant interaction between cultivars and years was detected, whereas no difference was observed between different years. This indicates that DI value differences among cultivars were consistently different from year to year.
Discussion
As cherry planting area in China continues to rise, P. circumscissa CLS increasingly represents a serious threat to the cherry industry and has been linked to substantial yield losses (Liu et al., 2012). However, there is no information regarding cherry cultivar resistance caused by P. circumscissa. The objective of this study was to evaluate the resistance of main cherry cultivars to guide breeding and disease management efforts.
The identification of CLS-resistant plants typically depends on field screening studies, which can be a useful and convenient approach (Maji et al., 2009; Olango et al., 2017; Zandjanakou-Tachin et al., 2013; Zhu et al., 2018). In our study, we observed significant variability in the ability of 52 cherry cultivars to resist CLS under field conditions, and grouped them into four different resistance categories based on DI values. However, cultivars that were immune or highly resistant were not observed among analyzed cultivars. Variable resistance values for the same cultivars were also observed in different years of field observation (Table 1), suggesting that resistance may depend on environmental conditions to some extent. Field resistance studies should be conducted over multiple years. To validate our results, data were collected from 3 years to provide reliable estimates of genetic resistance. Correlation coefficients varied from 0.65 to 0.69 when comparing pairs of DI values between years and were highly significant (Table 3), indicating that resistance was reasonably consistent from year to year.
Although field studies are a common approach to identifying disease-resistant germplasm resources, they are also influenced by environmental variations in factors such as humidity and temperature. In contrast to field assays, detached leaf assays can be conducted in a controlled setting and have been used previously to evaluate apple, mango, citrus, and other perennial fruit trees (Araujo et al., 2014; Francis et al., 2010; Yin et al., 2013). In our study, we used artificial inoculation assays to assess the resistance of 52 cherry cultivars to CLS. Consistent with several prior studies of disease resistance (Foolad et al., 2015), we obtained similar but not identical results under field conditions and controlled conditions.
In general, some varieties display resistance under field conditions but are susceptible under controlled conditions. In our experiment, ‘Jiahong’ was resistant in the field and susceptible in the detached leaf assay. A similar result was also observed for ‘Van’. Other types of inconsistencies in the results of these two assays were also observed. For example, ‘SYT1504’ and ‘SYT1506’ were found to exhibit moderate resistance in the field study but were found to be resistant in the detached leaf assay. The observed variations in DI values and ranking between our different assay methods are likely attributable to environmental variations. The high humidity that was maintained for all in vitro studies is generally conducive to the growth of fungi (Dorrance and Inglis, 1997; Michalska et al., 2011), potentially resulting in higher DI values in detached leaf assays relative to field observation.
We found that correlations between field observation study results and detached leaf assay results were strong (0.59–0.70; Table 4). An even stronger correlation was observed when comparing detached leaf assay results to the results from the average of all 3 years of field observation (r = 0.70, P < 0.01), confirming that true resistance to infection could be observed under field conditions over several years. Strong positive correlations between these two assays suggests that these approaches can be used reliably to screen for CLS resistance.
Analysis of variance assessment of the resistance of different cultivars to Passalora circumscissa cherry leaf spot based on field observations.
Detached leaf assays offer clear value as a tool for identifying CLS-resistant cherry cultivars, because these assays can be conducted rapidly and allow for the controlled screening of a large number of potentially resistant varieties. Our results further demonstrate that the results of these detached leaf assays correlate with field observation results, and should thus be used as a complementary strategy to expedite the selection of potentially or partially resistant cultivars that can then be evaluated in a more natural field setting.
To date, few studies have evaluated commercial cherry cultivars systematically to identify CLS-resistant varietals. In our study, we used both field observations and artificial inoculation approaches to screening for such CLS resistance among 52 different cherry cultivars, leading us to identify clear differences among cultivars concerning susceptibility to P. circumscissa–induced CLS. Our results offer new information that can help cherry breeders and producers as they work to prevent or manage CLS outbreaks.
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